VPM 201: Veterinary Bacteriology and Mycology 6-7/10/2010. LABORATORY 5a - ENTEROBACTERIACEAE

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1 VPM 201: Veterinary Bacteriology and Mycology 6-7/10/2010 LABORATORY 5a - ENTEROBACTERIACEAE A large family of gram-negative bacilli. They grow readily on common culture media. Organisms are separated (the differential part of this very useful selective and differential media) into two groups by the use of MacConkey agar (MAC); those that ferment lactose (LFs) and those that do not (NLFs). A. LAB EXERCISES 1. A swab of feces from a neonatal calf with diarrhea is provided. Use the swab to inoculate a BA and MAC plate. Label your plates for incubation overnight. Tomorrow you will examine your plates and refer to demonstration material to assist you with a presumptive identification. B. DEMONSTRATIONS 1. Examine the following cultures growing on BA and MAC plates. Note the color and colony type and record. Which are lactose fermenters? E. coli Klebsiella pneumoniae Proteus mirabilis Salmonella typhimurium

2 2. Observe and record the TSI agar reactions (see Lab Handbook, p ) provided. E. coli Salmonella typhimurium 3. Examine E. coli, K. pneumoniae and Proteus mirabilis inoculated into urea, indole and citrate media (see Laboratory Handbook, p. 7 and 10) and incubated for 24 hrs. 4. Examine the photomicrograph and prepared slide (under oil on microscope) of an India ink wet mount of K. pneumoniae for the presence of a capsule. 5. E. coli vaccines used in cows and sows. C. QUESTIONS AND DISCUSSIONS 1. What media would you use to isolate E. coli from diarrheic feces? Explain? Which other diarrhea-causing bacteria will grow on the media you selected? 2. Describe the growth of P. mirabilis on BA and MAC agar. Can you differentiate it from the colonies of S. typhimurium? 3. How do the TSI agar reactions of S. typhimurium differ from E. coli? 4. What specimens will you examine from a case of septicemia? a. Live animal b. Dead animal 5. Are members of Enterobacteriaceae generally susceptible to penicillin? 6. Why do oral antibiotics predispose to salmonellosis?

3 LABORATORY 5b ENTEROBACTERIACEAE and other INTESTINAL INFECTIONS A. THE GASTROINTESTINAL TRACT AS A MICROBIAL HABITAT The normal microbial flora is a host-defense barrier, the result of a host-parasite balance which has evolved over the millenia. The flora is remarkably stable. Particular microorganisms are adapted to each site along the gastrointestinal tract and occupy the optimal niche available to them. It is difficult for exogenous bacteria to establish because (1) they may have to compete with existing flora for mucosal receptors, (2) they may be inhibited by metabolic by-products, especially fatty acids, (3) they have to compete with existing flora adapted to the fierce competition for nutrients within the intestine. B. GASTROINTESTINAL MICROFLORA The gastrointestinal microflora is highly complex. In the large bowel of humans there are 8 about 500 species of bacteria at >10 bacteria per gram of content. The large bowel of animals contains 10 bacteria per gram - if it were one order higher feces would be solid bacteria. These bacteria are largely anaerobic, outnumbering facultative aerobes such as E. coli by 100 to 1. Many of these anaerobes are strict and most are nonpathogenic, lacking all virulence mechanisms. Many genera are found in the intestine, of which the most common are Bacteroides, Fusobacterium, Eubacterium, Peptostreptococcus and Bifidobacterium. These bacteria colonize in particular sites of the intestinal tract soon after birth, multiply to large numbers and remain at these numbers throughout life. There is evidence that a similarity exists between the antigenic determinants on the surface of mucosal intestinal epithelial cells and the indigenous microflora. These bacteria provide a stimulus for the development of the intestinal wall and of the immune system, as well as making major contributions to digestion in herbivores and preventing the establishment of certain pathogens. Perhaps one reason that diarrhea is so common in young animals is that indigenous flora have not become firmly established.

4 1. Mouth - The dominant bacteria are strict anaerobes including spirochetes, aerobic and anaerobic Actinomyces, and streptococci. Streptococci are important in the development of dental caries; spirochetes, Actinomyces viscosus, and Porphyromonas asaccharolytica (Bacteroides asaccharolyticus) are important in the development of periodontal disease. 2. Stomach - The acid ph of the stomach prevents the establishment of a bacterial flora in most animals. Many bacteria from the saliva are destroyed in the stomach. 3. Small Intestine - The upper small intestine has few bacteria present but numbers increase to about /gram of content in the lower ileum. In the upper bowel low ph and bile salts limit bacterial growth and peristaltic movements sweep bacteria down the tract. The flora of the lower small bowel consists of the anaerobes described above as well as, nonpathogenic E. coli, fecal enterococci and Clostridium perfringens present in moderate numbers. 4. Large Intestine - The colon and caecum have a massive bacterial flora, predominantly anaerobic, with a highly complex ecology. The fatty acid products (acetic, butyric) of anaerobic fermentation coupled with low ph and E h (oxidation-reduction potential) are toxic to members of the Enterobacteriaceae, such as Salmonella. Removing the anaerobic flora in mice by antibiotics can 6 reduce the infective dose of Salmonella from 10 to 1 bacterium. C. DISRUPTION OF THE INTESTINAL FLORA The protective role of intestinal microflora can be compromised by the following: 1. neonatal animals where the flora has not been fully established. 2. by antibiotics which select for resistant bacteria and for yeasts. Examples are Clostridium difficile colitis in humans or rabbits, Clostridium spiroforme in rabbits selected for by clindamycin and selection of Salmonella and Clostridium species in horses by tetracyclines. 3. stress can result in detectable changes in the large bowel flora. 4. by a change in diet that may produce minor changes in the large bowel flora.

5 D. PATHOGENIC MECHANISMS IN INTESTINAL DISEASE Most bacterial pathogens go through a two-stage process to initiate intestinal disease; firstly, by attaching to the target cell, and secondly, by producing a toxin or in some other way causing disease. Attachment is particularly important in the small bowel in order to overcome the washout effect of peristalsis (E. coli, V. cholerae, C. perfringens). Gastrointestinal pathogens cause disease in the small intestine, perhaps because there is less bacterial competition in this site. E. LABORATORY EXAMINATIONS 1. Direct Examination In general direct examination of fecal material is not a useful diagnostic procedure due to the high number of different types of bacteria present in the large bowel. Many will mimic the shape of pathogens. Examples include: nonpathogenic spirochetes in swine which are similar to Brachyspira hyodysenteriae, the large number of gram-negative rods which are morphologically similar to Salmonella and pathogenic E. coli etc. Exceptions include: clumps of acid-fast Mycobacterium avium subsp. paratuberculosis (Johne s disease), gentle jejunum mucosal scrapings of neonates with E. coli diarrhea will show a 10:1 ratio of bacteria to epithelial cells and in clostridial enterotoxemia large numbers of C. perfringens will be present. 2. Culture The main problem is to isolate specific bacterial pathogens against a background of a large number of non-pathogenic organisms. Generally, this is not a difficulty with E. coli because enteropathogenic types will predominate in disease. For Salmonella, however, we generally require a broth enrichment process. One example is Rappaport media (see Laboratory Handbook, p. 9) which is incubated for 24 hours before subculturing to another selective medium, Modified Semisolid Rappaport-Vassiliadis Medium (MSRV), (see Laboratory Handbook, p. 9). 6 Otherwise it would not be possible to recover 10 Salmonella/gram of feces 8 against a background of say 10 normal flora E. coli/gram. For Campylobacter

6 jejuni (one of the curved, gram-negative gastrointesintestinal pathogens) we use media with various antibiotics, such as Modified Preston (Campy) agar (see Laboratory Handbook, p. 7) for selective isolation. Anaerobic enriched blood agar with spectinomycin is used to isolate Brachyspira hyodysenteriae. 3. Biopsy and Histology Bacteria can sometimes be demonstrated in histological preparations of intestine; e.g. Lawsonia intracellularis in proliferative adenomatosis of swine and horses (less frequently), Brachyspira hyodysenteriae in swine dysentery, Clostridium perfringens in necrotic enteritis, E. coli in enteropathogenic E. coli diarrhea. F. LABORATORY EXERCISES Examine your BA and MAC plates inoculated yesterday with fecal-swabs from neonatal calves with diarrhea. Is there a predominant colony? If so what are the colony features? What is the gramstain and microscopic morphology like? Can you make a presumptive diagnosis? G. DEMONSTRATIONS 1. Enrichment isolation of Salmonella from feces of an adult horse with diarrhea using Rappaport broth and MSRV medium.

7 2. Examine a selective Campylobacter agar that was inoculated with feces from a puppy with a moderate, mucoid diarrhea and incubated microaerophilically (reduced oxygen) for o o 48 hours. This colony grew at 42 C but not at 25 C. It is also, catalase, oxidase, H S and hippurate positive. 2 Examine the gram-stain prepared from this culture. Note - these spiral/curved ( seagull-shaped ) pathogens are thin ( ìm) and stain very lightly with safranin. They can be challenging to see. Colony character: Gram morphology: Identity? Campylobacter Significance of this organism? 3. A silver-stained section from the ileum of a pig with proliferative intestinal adenomatosis. Locate the darkly stained, thin, curved bacteria making up microcolonies within the apical cytoplasm of crypt epithelial cells. What is most likely candidate for this pathogen? Vaccination is part of the management strategy for this important swine pathogen. 4. Examine the new Pathotyping/Virotyping PCR-based approach utilized by the E. coli reference laboratory at the Université de Montréal, Quebec.

8 H. QUESTIONS: 1. What serotypes of Salmonella are common pathogens in animals? 2. Why is Salmonella such a ubiquitous pathogen of animals? What factors predispose to infection? 3. Why is antibiotic resistance such a problem in Enterobacteriaceae such as Salmonella and E. coli? 4. Why is diarrhea so common in young animals? 5. How would you differentiate pathogenic E. coli from non-pathogenic E. coli after you have cultured a diarrheic fecal sample? 6. Will Salmonella grow at 42 C? 7. What specimens will you submit from calves with suspected E. coli diarrhea? a. Live animal: b. Dead animal: